Evaluation of Functional Selectivity of Haloperidol, Clozapine, and LASSBio-579, an Experimental Compound With Antipsychotic-Like Actions in Rodents, at G Protein and Arrestin Signaling Downstream of the Dopamine D2 Receptor.

LASSBio-579, an N-phenylpiperazine antipsychotic lead compound, has been previously reported as a D2 receptor (D2R) ligand with antipsychotic-like activities in rodent models of schizophrenia. In order to better understand the molecular mechanism of action of LASSBio-579 and of its main metabolite, LQFM 037, we decided to address the hypothesis of functional selectivity at the D2R. HEK-293T cells transiently coexpressing the human long isoform of D2 receptor (D2LR) and bioluminescence resonance energy transfer (BRET)-based biosensors were used. The antagonist activity was evaluated using different concentrations of the compounds in the presence of a submaximal concentration of dopamine (DA), after 5 and 20 min. For both signaling pathways, haloperidol, clozapine, and our compounds act as DA antagonists in a concentration-dependent manner, with haloperidol being by far the most potent, consistent with its nanomolar D2R affinity measured in binding assays. In our experimental conditions, only haloperidol presented a robust functional selectivity, being four- to fivefold more efficient for inhibiting translocation of ß-arrestin-2 (ß-arr2) than for antagonizing Gi activation. Present data are the first report on the effects of LASSBio-579 and LQFM 037 on the ß-arr2 signaling pathway and further illustrate that the functional activity could vary depending on the assay conditions and approaches used.

Synthesis and pharmacological evaluation of new N-phenylpiperazine derivatives designed as homologues of the antipsychotic lead compound LASSBio-579.

In an attempt to increase the affinity of our antipsychotic lead compound LASSBio-579 (1-((1-(4-chlorophenyl)-1H-pyrazol-4-yl)methyl)-4-phenylpiperazine; (2)) for the 5-HT(2A) receptor, we synthesized five new N-phenylpiperazine derivatives using a linear synthetic route and the homologation strategy. The binding profile of these compounds was evaluated for a series of dopaminergic, serotonergic and alpha-adrenergic receptors relevant for schizophrenia, using classical competition assays. Increasing the length of the spacer between the functional groups of (2) proved to be appropriated since the affinity of these compounds increased 3-10-fold for the 5-HT(2A) receptor, with no relevant change in the affinity for the D2-like and 5-HT(1A) receptors. A GTP-shift assay also indicated that the most promising derivative (1-(4-(1-(4-chlorophenyl)-1H-pyrazol-4-yl) butyl)-4-phenylpiperazine) (LASSBio-1635) (6) has the expected efficacy at the 5-HT(2A) receptors, acting as an antagonist. Intraperitoneal administration of (6) prevented apomorphine-induced climbing behavior and ketamine-induced hyperlocomotion in mice, in a dose dependent manner. Together, these results show that (6) could be considered as a new antipsychotic lead compound.

Biotransformation of LASSBio-579 and pharmacological evaluation of p-hydroxylated metabolite a N-phenylpiperazine antipsychotic lead compound.

Using a combination of docking and molecular dynamics simulations, we predicted that p-hydroxylation by CYP1A2 would be the main metabolic pathway for the 1-[1-(4-chlorophenyl)-1H-4pyrazolylmethyl] phenylhexahydropiperazine, LASSBio-579 (3). As the result of a screening process with strains of filamentous fungi, Cunninghamella echinulata ATCC 9244 was chosen to scale up the preparation of the p-hydroxylated metabolite (4). About 30 min after i.p. administration of (3) to rats was identified as the p-hydroxylated metabolite, confirming our in silico previsions. Chemical synthesis of the metabolite was performed and allowed its pharmacological evaluation in binding assays revealing its high affinity for D2 and D4 receptors, indicating that this metabolite should participate to the antipsychotic effect of (3) in vivo. Furthermore, we report here that both (3) and its p-hydroxylated metabolite (4) have a much lower affinity than clozapine for two receptors involved in adverse reactions. Voltammetric assays were useful to understand the redox profile of (3).